Membranes, both natural and synthetic, play critical roles in many fields. The utility of such membranes depends on a number of factors, physical, mechanical, chemical and biological. In many cases, of central issues are the barrier properties of the membrane, which determine the degree to and rate at which species of various types can pass through the membrane. It is often desirable to modify these barrier properties for the specific use. One of the most evident of natural membranes is skin.
Skin as a Barrier
The skin, the largest organ of the human body, has three layers—the epidermis, dermis and subcutis. The subcutis, the deepest layer, provides thermal insulation and has a shock-absorbing effect that helps protect the body's organs from injury. The dermis, the middle layer, contains hair shafts, sweat glands, blood vessels and nerves. The top layer of the skin is the epidermis, separated from the other layers of skin by the basement membrane which serves as the “glue” at the dermal-epidermal junction. The epidermis is relatively thin, and it is divided into four layers, from the innermost to outermost: the basal cell layer, stratum spinosum, stratum granulosum, and stratum corneum. The basal cell layer contains basal cells which divide and differentiate into other cells in the epidermis, and melanocytes, the cells that make melanin which gives skin its color. The stratum spinosum lies outside the basal cell layer and is comprised of keratinocytes, cells that make the protein keratin, an important component of the stratum corneum as well as of hair and nails. Cells in the stratum granulosum are flattened and contain dark granules that are expelled and provide the “cement” that holds cells together in the overlying stratum corneum. The stratum corneum, the outermost layer of the epidermis, is only some 20 μm thick, yet contributes over 80% to the skin permeability barrier. It is comprised of overlapping, flat corneocytes organized in columnar clusters; the dusters are sealed with multi-lamellar lipid sheets that are covalently attached to the cell membranes and are tightly packed. The stratum corneum is thicker in areas like the palms and soles that withstand more daily wear and tear than those of other parts of the body. The epidermis also contains Langerhans cells, which act as part of the skin's defense against infection.
Skin serves as the body's natural barrier against incursion of chemical or pathogenic factors, but it is a dynamic environment and there is major commercial interest in developing ways in which the barrier properties of skin can be modified.
Delivery through the Skin: A transdermal delivery route for therapeutics has major attractions compared with the oral route of administration as (i) it avoids first-pass liver metabolism of the drug, (ii) circumvents exposure of the drug to the chemical rigors of the gastrointestinal (“GI”) tract, (iii) may permit delivery of drugs with short biological half-lives and/or narrow therapeutic windows, (iv) may reduce adverse events in patients such as GI distress, (v) may offer more uniform plasma dosing of the drug, (vi) allows prompt interruption of dosing, and (vi) may increase patient compliance.
Active transdermal delivery routes, in which an external stimulus is applied to drive the drug through the barrier, include iontophoresis, sonophoresis, electroporation, microneedles, and application of high velocity solid particles [1] or liquids [2,3] (see [4]). These all require the application of a physical device, with irritation and compliance often an issue. A passive delivery route, in which a formulation containing the drug needs simply to be applied to the skin is substantially preferred. The prime requirements, in general, for a passive transdermal route are (I) that sufficient skin permeation can be achieved, (ii) that skin irritation and skin sensitization be avoided, and (ii) that reasonable delivery efficiency be accomplished.
Only a small number of drugs have been approved for application in transdermal patches (including scopolamine, nitroglycerin, clonidine, estradiol, nicotine, fentanyl, testosterone, norelgestromin with ethinyl estradiol). These share the three characteristics of (I) low molecular mass (<500 Da) [5], (ii) high lipophilicity, and (iii) small required dose (up to milligrams) [4].
Passive approaches for transdermal delivery of drugs based on vesicles, such as liposomes, have shown some promise for other classes of molecules [6-9]. However, these technologies have yet to appear in an FDA-approved transdermal patch product despite more than 20 years of work on vesicle-based formulations.
More than 300 chemical penetration enhancers (“CPEs”) have been considered in the literature [10]; [11] although few are useful in a practical sense—many do not provide a significant enhancement of transdermal drug permeation, and most cause skin irritation or present other safety issues. However, Karande et al. have discovered recently that rare combinations of CPEs, called SCOPE formulations, can cause pronounced permeability enhancement, yet little or no skin irritation [12]. A handful of SCOPE formulations were found amongst 5,040 binary CPE combinations. With more than 300 individual CPE's to consider, however, the space of binary and higher combinations is vast, so that very efficient methods for screening how CPE combinations affect skin barrier properties are desirable.
Transdermal delivery also has potential as a route for the delivery of proteins [13] and of genes into the body [12]. In order to develop chemical agents that are effective at promoting the permeation through skin of proteins or of deoxyribonucleic acid (“DNA”) or ribonucleic acid (“RNA”), what is again desirable are efficient experimental means of screening large numbers of combinations of such agents and many different packagings of the proteins or nucleic acids, for their effectiveness at achieving effective transport.
Delivery into the Skin: For dermatological indications and for cosmetic applications it may be desirable to delivery an active agent into the skin, but to avoid, if possible, passage into the serum. Local anesthetics [14] are a similar example. The skin is rich in antigen presenting cells, such as the Langerhans cells, so that a dermal Immunization route can be effective, although today required by needle or a jet injector, with the attendant issues of localized soreness, erythema, and hematoma at the injection site.
Avoidance of Penetration into or through the Skin: With wash, rinse and cleanser products, amongst, others, it is desirable that barrier properties of the skin not be impaired, to avoid ingress of potentially harmful agents. Cosmetic benefits from lipid formulations claimed to restore skin barrier have been reported [15].
Modification of Other Skin Barrier Properties: In several classes of medical devices electrical signals are sampled via surface mounted electrodes, such as in electrocardiography or external sphincter electromyography, for which modification of the skin electrical properties is desirable.
Altering the Sensory Properties of Skin: Many classes of personal care products, that include cosmetics, lotions, salves, creams, moisturizers, exfoliants, cleansers or colorants, improve the health, the feel or the appearance of skin.
Emollients, which soften skin, and moisturizers, which add moisture, are used to correct dryness and scaling of the skin. Dry skin results from loss of water from the stratum corneum, causing it to lose its flexibility and become cracked and scaly. The stratum corneum contains natural water-holding substances that retain water seeping out from the deeper layers of the skin. Water is also retained in the stratum corneum by a surface film of natural oil (sebum) and broken-down skin cells, which hinder trans-epidermal water loss (“TEWL”) through evaporation. Moisturizers and emollients can function through one or both of two actions [16]. Occlusives provide a layer of oil on the surface of the skin that slows water loss and thus increases the moisture content of the stratum corneum. Humectants are substances, exemplified by glycerin, urea or alpha hydroxy adds [17] such as lactic add or glycolic add, that, when introduced into the stratum corneum, increase its water holding capacity.
An exfoliant, or peeling agent, acts to slough away dead epidermal skin cells and encourage accelerated cell renewal, thus promoting soft and smooth skin that has visual appeal. Exfoliants function by promoting thinning of the stratum corneum through a descaling or keratolytic action.
To develop formulations that are effective as moisturizers, emollients or exfoliants, to assess the impact on skin of other topical applications such as cosmetics, sun screens, salves and cleansers, and to evaluate the biological impact of prospective active Ingredients in cosmetics, as in cosmeceuticals, what is needed is an efficient experimental means of screening large numbers of such formulations for their ability to do one or more of: (i) adsorb to the outer skin surface, (ii) be absorbed into the stratum corneum or other skin layers, (iii) permeate through the stratum corneum, (iv) permeate through the other skin layers and into the vasculature.
Skin Permeation Studies
The traditional method of performing skin permeation studies, including of topical and transdermal drug delivery formulations as well as of ophthalmics, cosmetics, skin care products and pesticides, employs a vertical diffusion cell, first described by T. Franz [18]. Permeation of a chemical agent from an upper donor well, through a skin sample, into a lower receptor well is assessed, under steady state conditions, through analysis of the concentration of chemical agent in the donor and receptor wells, such as by high performance liquid chromatography (“HPLC”). A single Franz diffusion cell can typically perform about one test per square inch of skin per day. While an automated Franz diffusion cell—HPLC system with 6 cells is now available from Logan Instruments Corporation of Somerset, N.J. (www.loganinstruments.com), use of a Franz cell requires (i) a relatively large area of skin, (ii) a substantial equilibration time, and (iii) substantial manual handling.
Discrete designs different from the Franz diffusion cell have also been disclosed, including Bronaugh's Flow Through Diffusion cell [19,20] and Moody's AIVDA system [21]; these also operate on the same principle of steady-state flux measurements. Despite their claimed advantages over Franz diffusion cells, however, their efficiency in screening enhancers is similar to that with Franz diffusion cells.
A related device used to measure the flow of metabolites across a membrane is the Ussing chamber, originally developed to measure the passage of water and sodium ions across short-circuited, isolated frog skin. Like the Franz diffusion cell, the Ussing Chamber consists of an upper donor chamber and a lower receptor chamber, with passage of a chemical agent through the membrane that separates the chambers being measured by analysis of the receptor well contents as described in a paper by Ussing [22], which is incorporated herein by reference. It differs, though, in being equipped to circulate and aerate the buffer solutions on donor and receptor sides, and to measure also the electrical potential across the membrane. Individual Ussing chambers are available, for example, from World Precision Instruments, of Sarasota, Fla. (www.wplinc.com). A 6-fold Ussing chamber arrangement is available from Dipl.-Ing. K. Mussler Scientific Instruments, of Aachen, Germany (www.kmsci.de). Ussing chambers or modified Ussing chambers (e.g. [23]) have been used extensively to measure ion and metabolite transport across many types of membrane but, like the Franz diffusion cell, the Ussing chamber is unsuitable for use in high throughput screening.
An alternative to these discrete cell designs is to use an array format. Thus, U.S. Pat. No. 5,490,415 [24], which is incorporated herein by reference, describes an apparatus used to test diffusion of a drug through a test membrane in which a number of open-top receptor vessels addresses a test membrane captured between this receptor vessel array and a mirror-image donor vessel array. The drug diffuses from a given donor well through the test membrane and into a receptor liquid in the corresponding receptor well. Samples of the receptor liquid might then be transferred using a programmed liquid transfer system, perhaps for assay by a scintillation counter. U.S. Pat. No. 6,043,027 [25], which is incorporated herein by reference, describes testing devices, systems, and methods for evaluating the permeation of various chemicals through different types of cells. One such device is described to comprise a base member and a top member having a plurality of wells which are aligned when the top member is secured to the base member. A membrane sheet which includes at least one layer of cells grown on the sheet is placed between the base member and the top member prior to assembly. Test samples are placed into the wells in the top member and samples are removed from the top and bottom wells at a later time and tested to determine the amount of test sample which permeated through the cells [25].
Still more recently, WO 02/06518 A1 [26], which is incorporated herein by reference, claims an apparatus for measuring transfer of components across a tissue, comprising a support plate; an array of samples supported by the support plate; a tissue specimen overlaying the array of samples; and a reservoir plate secured to a side of the tissue specimen opposite the array of samples, the reservoir plate having an array of reservoirs [26]. Cima et al. recognized the need for a suitable means to fill donor and/or receptor wells. WO 02/06518 A1 [26] claims a feed canula, having a sample feed source and an air evacuation space, which punctures a rubber septum which covers one side of a donor well. By placing the tip of the canula on the tissue it is claimed that air in the donor well will be forced out of the donor well into the air evacuation space, eliminating any air pockets adjacent to the tissue. It is claimed that the tip of the canula can be progressively retracted toward the septum as donor well filling proceeds (as otherwise the air evacuation space will fill with donor well contents). However, this method requires contact of the sharp tip of the canula with the tissue, potentially causing damage to the barrier layers on the top of the tissue. Further, without sophisticated methods it is difficult both to determine to precisely what depth the canula must be inserted (leading to the possibility of severe tissue barrier damage and, minimally, to uncertainty in each case whether or not such damage has occurred), and the extent to which well filling has progressed (making concerted retraction of the canula difficult to control). Further, this ‘from near the bottom Introduction’ method is not effective in practice at eliminating bubbles, particularly for viscous samples. Additionally, this approach is not claimed to be useful in achieving complete filling of a well compartment.
What was termed a combinatorial method for rapid screening of drug delivery formulations has been disclosed in works by Mitragotri et al. [27] and Karande et al. [28], both of which are incorporated herein by reference. One embodiment of the system described by Mitragotri uses of an array of wells, each potentially containing a different formulation, applied to a single piece of skin, with permeation being monitored via quantitative changes in the single point conductivity of the stratum corneum in the vicinity of each well. Skin conductivity measurements provide a rapid assay to determine the effect of enhancers on skin permeability [28]. The conductivity measurements may be calibrated by comparison with direct permeation measurements, either in the same experimental set-up or in Franz diffusion cells operated under similar conditions [28].
Systems providing parallel diffusion cells have the potential to provide significant gains in the speed with which permeation measurements can be made. However, in general, techniques have not yet been developed for such approaches that (i) are suitable for making measurements at short contact times between skin and formulations (ii) provide automation-friendly methods for ensuring contact of donor and receptor fluids with skin by avoiding the presence of bubbles (iii) allow for partial or complete inversion of the apparatus, and (iv) provide support measurements of skin properties other than permeation.
Unmet Needs
To be able to efficiently asses the effect on the barrier properties of a membrane of a test formulation, suitable for application in high throughput, there is a need for methods and apparatus that would desirably have the following characteristics:                (1) be able to accommodate measurements on skin, as well as on a broad range of other natural and synthetic membranes;        (2) require minimal amount of membrane material and reagents per measurement;        (3) support direct measurements of the permeation, preferably of molecular and particulate entities;        (4) accommodate a range of formulation types, encompassing aqueous or non-aqueous solutions, emulsions and hydrocarbon-based lotions, formulations that might be rubbed onto the skin, and formulations with a volatile component that will evaporate.        (5) be compatible with automation, robotics, experiment and data management systems and suitable for integration into a high throughput experimentation workflow;        (6) support both direct and indirect measurements of the electrical response of the stratum corneum, over a range of times of contact of the test formulation(s) with the membrane, from less than a minute to many hours, and with the possibility of first measurements being accumulated within a few seconds of first test formulation contact;        (7) support measurements of each absorption, that is the degree to which a molecule or material is taken up by the stratum corneum, but without permeation into or through the epidermis, adsorption, that is the adherence of molecular or other entities to the skin exterior, and exfoliation, that is the extent of sloughing off of material from the stratum corneum;        